US6995133B1 - Utilization of interferon alpha 5 in the treatment of viral hepatopathies - Google Patents

Utilization of interferon alpha 5 in the treatment of viral hepatopathies Download PDF

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US6995133B1
US6995133B1 US09/674,445 US67444500A US6995133B1 US 6995133 B1 US6995133 B1 US 6995133B1 US 67444500 A US67444500 A US 67444500A US 6995133 B1 US6995133 B1 US 6995133B1
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ifnα
liver
interferon
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Jesús Prieto Valtueña
Ma Pilar Civeira Murillo
Esther Larrea Leoz
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/212—IFN-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the production of interferon alpha 5 for use in compositions useful in the treatment of liver diseases of viral origin.
  • IFN-alpha 5 is the sole subtype of alpha interferon produced in the healthy liver and that its levels are clearly reduced in chronic hepatitis C, which suggests that this substance may be of therapeutic value in the treatment of this disease and other forms of viral hepatitis. Knowing the coding gene sequence for this interferon, its production through recombinant DNA technology in different hosts makes it possible to develop effective drugs for the treatment of liver diseases of this type at their different stages of development.
  • IFN ⁇ and IFN ⁇ type I interferon
  • IFN ⁇ is a family of thirteen polypeptides (subtypes) coded by different genes. IFN ⁇ is a glycoprotein produced by a single gene. Different cell types produce both IFN ⁇ and IFN ⁇ (1, 2).
  • Viral infection is the main stimulus for the production of type I interferon, although there are other factors which can increase its synthesis, such as bacterial components, double chain RNA, growth factors and other cytokines (1).
  • IFN ⁇ can interact with certain cytokines and with T cells regulating the growth and differentiation of the cells in the immune system (3).
  • IFN ⁇ genes are expressed as a matter of course in human tissue in healthy individuals (4), while the expression of particular subtypes is restricted to certain cell types (5, 6).
  • the induction of IFN by viruses is mainly regulated at transcription level. The specific activation of transcription occurs through the interaction of cell factors induced by viruses with the domains regulating the promoters of IFN ⁇ genes (7).
  • IFN ⁇ and IFN ⁇ subtypes have a common receptor at the cell surface.
  • Competitive binding tests at the receptor for different IFN ⁇ subtypes indicate that all of these combine at the same receptor, but with different affinities (8).
  • the biological activity of the different subtypes of IFN ⁇ is little known.
  • the IFN ⁇ 5 and IFN ⁇ 8 interferon subtypes appear to be those having the greatest antiviral activity.
  • Antiproliferative response also differs between the different subtypes (9).
  • unstimulated peripheral blood mononuclear cells express different IFN ⁇ subtypes (10).
  • IFN interleukin-12
  • monocytes infected by human immunodeficiency virus (11) A common mechanism for the persistence of viral infection is avoidance of the IFN system.
  • Many viruses have developed strategies to avoid the antiviral effects of IFN. Specifically, a selective defect in the production of IFN ⁇ has been described in monocytes infected by human immunodeficiency virus (11).
  • Hepatitis C virus is a single chain RNA virus which results in chronic infection in more than two thirds of persons infected.
  • the prevalence of infection by HCV is around 2 to 3% in the population of the West.
  • Studies performed in Europe show that 33% of patients with chronic HCV infection develop cirrhosis in a mean period of less than 20 years (12).
  • a significant proportion of these patients develop liver cancer, with an annual incidence of 1.4% (13).
  • the high rate of mutations in the virus and the production of a predominant profile of Th2 cytokines in comparison with Th1 have been described as being responsible for this high level of persistence by the infection.
  • Treatment with IFN induces a sustained response in around 30% of patients with chronic hepatitis C. The mechanism responsible for response or non-response to treatment with IFN is little understood.
  • the IFN system has only been studied in chronic HCV infection. There is no appropriate animal model for chronic HCV infection, and, because of this, investigations performed on humans are the only source of information on the pathophysiology and pathogenesis of chronic hepatitis C.
  • This invention describes the expression of IFN ⁇ and IFN ⁇ genes in the liver and in the peripheral blood mononuclear cells (PBMC) in healthy controls and patients with chronic hepatitis C.
  • PBMC peripheral blood mononuclear cells
  • IFN ⁇ subtype expressed in normal liver tissue and the liver tissue of patients with chronic hepatitis C. Expression of the different IFN ⁇ subtypes has also been analysed in PBMC in healthy controls and patients with chronic hepatitis C.
  • FIGS. 1 A–B Expression of alpha interferon/ ⁇ -actin RNAm (ordinate) in peripheral blood mononuclear cells (A) and in the liver (B) of healthy controls and patients with chronic hepatitis C (HCV-RNA+) (abscissa).
  • FIGS. 2 A–B Expression of beta interferon/ ⁇ -actin RNAm (ordinate) in peripheral blood mononuclear cells (A) and in the liver (B) of healthy controls (C) and patients with chronic hepatitis C (HCV-RNA+) (abscissa).
  • FIGS. 3 A–B Relationship between the initial quantity of total RNA (abscissa) and the strength of the PCR product band obtained by amplifying the RNAm of IFN ⁇ (•), IFN ⁇ ( ⁇ ) and ⁇ -actin ( ⁇ ) (ordinate, as counts ⁇ mm 2 ) in PBMC (A) and liver (B) samples.
  • IFN ⁇ and IFN ⁇ genes were analysed in samples from liver biopsies from 16 patients with chronic hepatitis C (9 men and 7 women, age range 24 to 71 years). Five of these patients showed cirrhosis. The viral genotype was determined in 14 patients and was 1b in 10 patients, 1a in 2 patients and genotype 3 in 1 patient.
  • IFN ⁇ and IFN ⁇ genes were determined in 12 samples of normal liver obtained by laparotomy from 12 control patients (9 men and 3 women, age range 49 to 70 years).
  • the laparotomies were performed on account of the presence of digestive tumours in 10 patients (4 colo-rectal, 5 gastric and 1 pancreatic) due to chronic pancreatitis in 1 patient and the presence of a hydatid cyst in another patient.
  • Liver histology was normal in the twelve cases. None of these control cases had received treatment before the liver sample was obtained.
  • mRNA levels of IFN ⁇ and IFN ⁇ were also determined in PBMC in 25 patients with chronic hepatitis C (14 men and 11 women, age range 24 to 69 years) (four of these patients had cirrhosis) and in PBMC from 23 healthy controls (10 men and 13 women, age range from 25 to 66 years).
  • the viral genotype for these patients was 1b in 22 patients, 1a in two patients and 3 in 1 patient.
  • the diagnosis of chronic hepatitis C was based on an increase in serum transaminases lasting more than 6 months, a positive result for anti-HCV antibodies (2nd generation ELISA, Ortho Diagnostic System, Raritan, N.J., USA), the presence of C virus RNA in serum (reverse-reaction transcription in the polymerase chain), and histological evidence of chronic hepatitis.
  • the severity of liver damage was evaluated using the Knodell index (16). Other causes of chronic hepatitis other than hepatitis C virus were ruled out. None of the patients had received treatment with IFN ⁇ during at least 6 months prior to the study.
  • liver samples were obtained by liver biopsy using a Tru-Cut biopsy needle (Baxter, Deerfield, Ill.). One third of the sample was immediately frozen in liquid nitrogen and kept at ⁇ 80° C. until total RNA extraction took place. The remainder of the sample was used for the histological investigation.
  • PBMC peripheral blood mononuclear cells
  • the serum samples were obtained by centrifuging from venous blood collected in sterile tubes. The serum was kept at ⁇ 40° C. until use.
  • RNA levels of IFN ⁇ and IFN ⁇ were determined using a quantitative polymerase chain reaction reverse transcription (RT-PCR) method using a thermocycler (Perkin-Elmer Gene Amp PCR system 2400).
  • RT-PCR quantitative polymerase chain reaction reverse transcription
  • thermocycler Perkin-Elmer Gene Amp PCR system 2400.
  • RNA from both the liver and PBMC
  • DNAse I amplification grade Gibco-BRL, Gaithersburg, Md., USA
  • the presence of traces of DNA was checked by including control reactions without reverse transcription. This step is required because of the absence of introns in IFN ⁇ and IFN ⁇ genes (18), which made it impossible for us to distinguish the product of PCR from the RNA or possible contaminating DNA.
  • a fragment of ⁇ -actin cDNA was amplified using a 10 ⁇ l aliquot of the cDNA obtained previously.
  • the IFN ⁇ was amplified by performing 30 or 33 cycles (PBMC or liver respectively) (94° C., 60° C. and 72° C. during 20, 15 and 30 seconds for each step respectively), the INF ⁇ was amplified by performing 30 or 35 cycles (PBMC or liver respectively) (94° C., 58° C. and 72° C. for 20, 15 and 30 seconds for each step respectively) and ⁇ -actin was amplified by reacting 18 or 25 cycles (PBMC or liver respectively) (94° C., 55° C. and 72° C.
  • oligonucleotides (5′-3′) d(TCCATGAGATGATCCAGCAG) (SEQ ID NO:2) and d(ATTTCTGCTCTGACAACCTCCC) (SEQ ID NO:3) were used as direction and antidirection primers respectively to amplify a fragment of 274 pairs of bases located between nucleotides 240–514 in the human IFN ⁇ gene (19). These oligonucleotides are direction primers designed to amplify all the subtypes of IFN ⁇ .
  • the oligonucleotides D(TCTAGCACTGGCTGGAATGAG) (SEQ ID NO:4) and d(GTTTCGGAGGTAACCTGTAAG) (SEQ ID NO:5) were the primers used to amplify a fragment of 276 base pairs located between nucleotides 349–625 of cDNA of human IFN ⁇ (20) d(TCTACAATGAGCTGCGTGTG) (SEQ ID NO:6) and d(GGTGAGGATCTTCATGAGGT) (SEQ ID NO:7) were the primers used to amplify a fragment of 314 base pairs (nucleotides 1319–2079) of the ⁇ -actin gene (21).
  • Validation curves for the PCR technique were prepared using known quantities of total RNA (from 0 up to 1 ⁇ g). As will be seen in FIG. 3 , with the total initial RNA quantities used for IFN ⁇ , IFN ⁇ and ⁇ -actin (0.5 ⁇ g, for both the liver and PBMC), we were within the linear range of the PCR amplification curve. The inter-test coefficient of variance for IFN ⁇ / ⁇ -actin was 22% and for IFN ⁇ / ⁇ -actin it was 24%. The identity of the PCR product obtained was checked for IFN ⁇ and IFN ⁇ by automatic sequencing (ABI prismTM 310 genetic analyser, Perkin Elmer).
  • RNA extraction, reverse transcription and the PCR reaction were performed as described above, using the IFN ⁇ direction primers mentioned.
  • the PCR product obtained was cloned using the commercial TOPO TA cloning kit (Invitrogen, Leek, Holland). At least 6 clones from each insert were sequenced in an automatic ABI PRISM 310 sequencer (Perkin Elmer, Foster, Calif.), using the Dye Rhodamine Terminator Cycle Sequencing Kit (Perkin Elmer, Foster, Calif.).
  • C virus RNA was determined using the RT-PCR technique (14, 22), using 2 pairs of specific primers for the non-coding 5′ region of the C virus genome.
  • the C virus RNA was quantified using the competitive PCR technique previously described by our (22).
  • the viral genotype was determined using Viazov's method (23) as already described previously (22, 24).
  • the test 5′G(R)CCGTCTTGGGGCC(M)AAATGAT (SEQ ID NO:8) was used to determine genotype 4.
  • the IFN ⁇ and IFN ⁇ results are presented as mean ⁇ standard error.
  • the normality of the variables was studied using the Shapiro-Wilks test.
  • Statistical analysis of IFN ⁇ and IFN ⁇ values in PBMC or liver was performed using non-parametric tests (Mann-Whitney U test) or parametric tests (Student's T). The association between quantitative variables was investigated using the Pearson or Spearman correlation coefficient, as appropriate. Windows SPSS 6.0 program was used for the statistical analysis.
  • the cDNA which codes for interferon- ⁇ 5 is cloned in pET14b vector (available commercially from Novagen).
  • This vector provides a sequence which codes for a series of histidine residues (1 kDa) which are translated in phase with the cloned cDNA to yield a merged protein which includes a 1 kDa histidine tail at its terminal amine end and then interferon- ⁇ 5, with a site between the two which can be cut by thrombin.
  • competent bacteria of the BL21 (DE3) strain are prepared, as this strain contains a gene which can be induced by T7 RNA polymerase, which is a necessary requirement for the subsequent production of protein.
  • the competent bacteria are converted with the vector previously obtained (pET14b with the cloned interferon- ⁇ 5 cDNA).
  • the transformed bacteria are selected by their growth in LB medium with ampicillin, as the vector contains a gene which is resistant to this antibiotic.
  • the transformed bacteria are grown in LB medium with ampicillin at 37° C. until an optical density of 0.4 at 600 nm is obtained. Then expression of the recombinant protein with IPTG is induced at a final concentration of 0.5 mM. In this way the lac promoter is induced and as a consequence the T7 RNA polymerase prometer which contains the vector and which regulates the expression of the cloned cDNA is induced. The culture is grown for a further 4 hours under the same conditions.
  • the precipitated bacteria are resuspended in 10 mM Tris/HCl buffer, 10% saccharose, 2 mM 2-mercaptoethanol and protease inhibitors. Homogenization was performed ultrasonically by incubation for 30 minutes with lysozyme at 4° C. This breaks down the bacterial wall and improves the yield of the extraction process.
  • the cytosol extract is obtained by centrifuging the homogenate at 100,000 g for 90 minutes. Protein production is checked by analysing the cytosol fraction by SDS-PAGE.
  • His-interferon- ⁇ 5 merged protein is purified by chromatography of the cytosol extract in a 2 ml nickel column. The protein is eluted by washing the column with 1 M imidazole. The pure protein is processed with thrombin and the interferon- ⁇ 5 is subsequently repurified by molecular exclusion chromatography.
  • the cDNA which codes for interferon- ⁇ 5 is cloned in an Agrobacterium tumefaciens expression vector.
  • This vector contains the potato promoter (the most abundant protein in the Solanum tuberosum tubercle), as well as a sequence which codes for a series of histidine residues (1 kDa) and which are translated in phase with the cloned cDNA to yield a merged protein which contains a 1 kDa histidine tail at its terminal amine end followed by interferon- ⁇ 5, with a site between the two which can be cut by thrombin.
  • competent bacteria of the GV2260 strain of Agrobacterium tumefaciens are prepared.
  • the competent bacteria are transformed using the previously obtained vector.
  • the transformed bacteria are selected by growth in LB medium with kanamycin, as the vector contains a gene which is resistant to that antibiotic.
  • Total protein extraction is performed from tubercles of the transgenic plants which express the interferon- ⁇ 5.
  • the purification of His-interferon- ⁇ 5 merged protein is carried out by chromatography of the protein extract obtained on a 2 ml nickel column.
  • the protein is eluted by washing the column with 1 M imidazole.
  • the pure protein is processed with thrombin and the interferon- ⁇ 5 is subsequently repurified using molecular exclusion chromatography.
  • the mRNA of the IFN ⁇ was amplified using universal primers for all the IFN ⁇ subtypes. The PCR amplification products were then cloned and sequenced. 41 clones from 4 different normal livers were analysed and we observed that the IFN ⁇ sequence in the 41 clones was the same and corresponded to the IFN ⁇ 5 subtype (Table 1). These results show that IFN ⁇ 5 is the only IFN ⁇ subtype expressed in normal liver.
  • the partial cDNA sequence of the IFN ⁇ 5 obtained from all the clones was shown to be SEQ ID NO: 1.
  • the PBMC from patients with chronic hepatitis C expressed IFN ⁇ 21, IFN ⁇ 5 and IFN ⁇ 7 (5, 12, and 1 clones respectively).
  • IFN ⁇ 21, IFN ⁇ 17 and IFN ⁇ 1/13 8, 1 and 2 clones respectively.
  • the mRNA levels of IFN ⁇ were determined using the semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) technique using universal primers to amplify all the IFN ⁇ subtypes. The values are expressed as the ratio of IFN ⁇ mRNA to ⁇ -actin mRNA.
  • IFN ⁇ 5 is the only IFN ⁇ subtype detected in normal liver, while a mixture of subtypes is observed in the liver tissue of patients with chronic hepatitis C.
  • Our findings indicate that in infection by HCV there is a marked reduction in the expression of the IFN ⁇ subtype normally expressed in liver tissue.
  • liver IFN ⁇ IFN ⁇ 5
  • IFN ⁇ 5 liver IFN ⁇
  • IFN ⁇ the second majority form of type 1 interferon, is a glycoprotein produced by a single gene.
  • transcription of the IFN ⁇ and IFN ⁇ genes is activated or repressed by various mechanisms (15).
  • HCV causes IFN ⁇ to be repressed in the liver, the expression of IFN ⁇ is increased in both the liver and PBMC. This indicates that VHC modulates the different type I IFN genes in the liver in a different way, and blocks the production of IFN ⁇ to permit the overexpression of IFN ⁇ .

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FR2824333B1 (fr) * 2001-05-03 2003-08-08 Genodyssee Nouveaux polynucleotides et polypeptides de l'ifn alpha 5
ES2303492A1 (es) * 2004-08-18 2008-08-01 Instituto Cientifico Y Tecnologico De Navarra S.A. Uso del factor de crecimiento semejante a la insulina tipo i y de interferon alfa en el tratamiento de una enfermedad hepatica cronica, kit y composiciones que los comprenden.
WO2006032711A1 (es) * 2004-08-18 2006-03-30 Instituto Científico Y Tecnológico De Navarra S.A. Uso del factor de crecimiento semejante a la insulina tipo i y de interferón alfa en el tratamiento de una enfermedad hepática crónica, kit y composiciones que los comprenden
KR101858598B1 (ko) 2010-02-01 2018-05-16 디그나 비오테크, 에스.엘. 인터페론 알파 5의 제조방법
CN103732242A (zh) * 2011-06-23 2014-04-16 迪格纳生物技术公司 用与IFN-α2b组合的IFN-α5在患者群体中治疗慢性丙型肝炎
LT6164B (lt) 2013-10-15 2015-06-25 Uab Biotechnologinės Farmacijos Centras "Biotechpharma" Sulieti interferono-alfa 5 baltymai su kitu citokinu ir jų gamybos būdas

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